DE2925534A1 - METHOD AND REAGENT FOR DETERMINING FRUCTOSE - Google Patents

Abstract

1. Process for the determination of fructose in the presence of other sugars or polysaccharides present in excess and for the determination of fructose-containing glycosides, especially in the presence of other sugars or polysaccharides present in excess, characterized in that the fructose, possibly first liberated from the glycoside, is reacted with UDPG and saccharose synthetase with the formation of UDP which is determined in known manner.

Description

Method and reagent for the determination of fructose.

The invention relates to a method and a reagent for the determination of fructose and fructose-containing glycosides, especially in the presence of other carbohydrates in excess.

The determination of carbohydrates belongs in food chemistry to the elementary analytical tasks. There are a large number of analytical methods which can be used to determine the carbohydrate composition. A summary can be found in "Handbuch der Lebensmittelchemie" II / 2 (1967), Springer-Verlag. However, the known analytical methods have the disadvantage of a very complex and cumbersome implementation, if it is not just the sum. of all carbohydrates - mostly via physical or

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physico-chemical process - to be determined (ζ. Β. extract content via refractometry, density determination or polarimetry), but also a selectivation of the to strive for different carbohydrate components.

The most important sugars in food chemistry are glucose, fructose and sucrose. Because of the lack of Chemical methods for the determination of glucose and fructose side by side is used in the field of food chemistry mostly the sum of both sugars is determined and this measured value is called "reducing sugar" or "reducing sugar" Sugar "is used to assess food. There are many expressions of this analytical weakness Food laws and regulations in which precisely these forms of sugar are anchored (wine law, fruit juice regulation, Regulation on fruit products).

In many cases, the determination of the extract content (determined via drying loss) is quite representative for the content of reducing sugars, or the Luff / Schoorl method is used (Chem. Weekbl. 9_, 678, 706 (1912); ZUL Sl_, 566 ( 1929)), with which one can evaluate the reducing properties of the monosaccharides to determine the content.

However, this process is very susceptible to failure if there are also other reducing components in the food present (vitamin C in fruit juices, galacturonic acid, Reductones in wine; see G. Baumann and K. Gierschner, Ind. Obst- und Gemüseverwertung ^ 6_, 165 (1971)). In in such cases a specific determination of glucose and fructose becomes indispensable. True are also Methods known to be able to make such a differentiation (e.g. alkaline iodine method), but ' are these prone to failure.

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The use of an enzymatic method, which has become more and more widespread in recent years. It is based on the phosphorylation of fructose with ATP in the presence of hexokinase and phosphoglucose isomerase and formation of glucose-6-phosphate and its Determination with its dehydrogenase and NADP. But even this method can only be used in its simple form if when the concentrations of glucose and fructose are in ratios of 1: 0.03 to 1: 30. Concentration ratios lying outside this range are also compelling when using the enzymatic one Methodology for applying complementary techniques, e.g. removing a sugar component. Excess In addition to small amounts of fructose, glucose can be completely removed with the help of glucose oxidase (GOD) that the subsequent measurement of fructose is possible (H. U. Bergmeyer, "Methods of enzymatic analysis" 1974, Verlag-Chemie, Weinheim, p. 1349). With extreme excesses of glucose in addition to low fructose concentrations on the other hand, a complete removal of the glucose is not possible (H. 0. Beutler, G. Michal, G. Beinstingl: German Lebensm. Rundschau 1978). In these cases, the glucose concentration when using the GOD method can only be reduced to a residual concentration, after which a measurable concentration of both sugar components results.

The determination of the content of other carbohydrates containing fructose units, such as. B. sucrose. (GlucosyIfructose) causes trouble, though there are unfavorable sugar ratios. In general, sucrose can be obtained by a polarimetric method be determined with sufficient specificity (optical rotation value of a solution before and after inversion). This method but fails in the presence of high concentrations

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other sugars or from polysaccharides. So it is not possible to use this method to obtain small sucrose concentrations in addition to high levels of starch in food for children or dietetic foods to determine. Here the enzymatic method would be below Use of ß-fructosidase for sucrose measurement be suitable. This procedure, in turn, is not very applicable, if the food also contains high concentrations of malt extract and glucose. In the case of unfavorable Ratios of sucrose and other carbohydrates must therefore - even when using enzymatic Measurement techniques - an extensive sample preparation precedes before the sucrose can be determined.

In clinical chemistry, the determination of the fructose concentration is used for certain diagnostic purposes to be useful. Since this concentration in the blood is very low compared to the simultaneously high concentration of If glucose is present, this concentration can only be measured with great effort.

For inulin clearance, the determination of inulin in the blood is desirable. After hydrolysis of the inulin (polyfructose, Fructosan) acid treatment or inulinase results in the hydrolysis product fructose, from its concentration on the inulin content can be concluded.

The invention is therefore based on the object of creating a method for determining fructose which does not have the above disadvantages and especially in the presence of other carbohydrates in excess, like sugars or polysaccharides. This procedure is also intended for the determination of fructose released from other carbohydrates be suitable and thus the determination of such fructose-

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containing carbohydrates.

According to the invention, this object is achieved by a method for determining fructose and fructose-containing substances Glycosides, especially in the presence of other sugars or polysaccharides in excess, which is characterized in that the optionally first set free from the glycoside Fructose is reacted with UDPG and sucrose synthetase to form UDP, which is known per se Way is determined.

Surprisingly, according to the invention, it is possible to obtain fructose and to determine fructose-containing glycosides independently of the concentration of other sugars or carbohydrates. For example, it has been found that cellulose, pentosans, hemicelluloses, Mannans, galactans, inulin, levan, raffinose, sucrose, lactose, maltose, glucose phosphate, mannose, mannose phosphate, Mannitol, mannitol phosphate, galactose, galactose phosphate, fructose phosphate, sorbitol, sorbitol phosphate, Inosite, pentoses such as ribose, arabinose and xylose, pentose phosphate, erythritol and erythritol phosphate are not disturb. This is all the more surprising as, according to the literature, other carbohydrates, such as. B. Fructose-6-phosphate, converted by sucrose synthetase. It is just as astonishing that the fructose determination is not disturbed by a high excess of sucrose, which is a reaction product.

In addition, it was by no means foreseeable that the sucrose synthetase would even be used for the determination of fructose would be suitable, especially since this enzyme has been known for more than 20 years without any such use has considered so far.

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Various methods can be considered for measuring the UDP formed in addition to sucrose in the reaction. The reaction, known per se, of UDP with phosphoenolpyruvate (PEP) in the presence of pyruvate kinase is preferred (PK) and lactate dehydrogenase (LDH) as well as NADH with the formation of NAD. This process can go through the following reaction equations illustrate:

PTf

( 1) UDP + PEP -> UTP + pyruvate

(2) Pyruvate + NADH + H ⁺ £ 122 ^ lactate + NAD ⁺

The photometrically measurable conversion of NADH to NAD is proportional to the fructose concentration. Other possibilities The determination of UDP is based on phosphorylation with a phosphate donor, such as ATP, in the presence the corresponding kinase and measurement of the dephosphorylated phosphate donor, such as ADP, according to known methods. Formed UTP can also be measured.

Another embodiment based on this principle consists in phosphorylation with formyl phosphate in the presence of formate kinase (E.C. 2.7.2.6) with the formation of formic acid, which in turn, for example with formate dehydrogenase (E.C 1.2.1.2) in the presence of NAD can be converted to form NADH, which is measured in a customary manner.

Another example based on this principle is the phosphorylation with arginyl phosphate in Presence of arginine kinase (E.C.2.7.3.3), cleavage of the formed free arginine with arginase (E.C 3.5.3.1) with the formation of urea, which is determined in the usual way is, for example, by cleavage with urease E.C 3.5.1.5) with the formation of ammonia, which is measured will.

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The inventive. Procedure is as with enzymatic Determination methods customary, carried out in buffered aqueous solution. In general it turns out to be a pH range between 6 and 10.5 is suitable. There are no restrictions on the type of buffer. Glycine buffer, glycylglycine buffer, Tris buffer and TRA buffer are preferred. Furthermore, the implementation expediently made in the presence of magnesium ions, for example in the presence of magnesium chloride.

As already mentioned, the one according to the invention is suitable Method not only for the determination of fructose itself, but also for the determination of others containing fructose Carbohydrates from which the fructose can be split off quantitatively, so that the released Fructose is a measure of the amount of carbohydrate. Such carbohydrates include, for example Disaccharides, trisaccharides, tetrasaccharides, pentaoses and polysaccharides. The following table shows examples of such fructose units containing Carbohydrates listed with details of their structure, enzymes suitable for cleavage and occurrence. So far no specific cleavage enzymes are available, the cleavage can take place chemically, for example by acid hydrolysis according to customary methods. An example this is done by hydrolysis with dilute perchloric acid.

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Surname structure Disaccharides Xylosyl fructose XyI Fru Furanosis GIu 1/3 Fru Maltulose o * -Glu 4 fru Cellobiulose ß GIu 4 Fru Palatinosis GIu 6 Fru Galsucrose Gal-3-Fru O Lactulose Gal-4-Fru <*> Planteobiosis Gal-6-Fru O
CD Fructosyl deoxy CD glucose For 1/6 GIu "^ Inulobiosis Fru 1/2 Fru O Trisaccharides revenues GIu 3/4 GIu 1/2 fru

Glucosyl sucrose
Gentianosis

GIu 3/6 GIu 1/2 Fru GIu 1/2 GIu 1/2 Fru

Umbelliferosis
Lactosucrose

Raffinose

Gal .1 / 2 GlU 1/2 Fru Gal .1 / 4 GIu .1 / 2 Fru Gal] / 6 GIu 1/2 Fru

Cleavage

onzyniü

Vox'koiiuuen

Bee honey

, Beer

^ -Glucosidase

Emulsin

Galactosidase

drug

Tobacco, corn

Invertase (yeast)

Invertase (yeast)

supplies gentiobiosis

and fructose

Emulsin

provides sucrose

and glucose

ro cn cn co

Invertase

Molasses. Extracts from sugar beet; disturbs
Crystallization of the
¹ ■ rrharose

Surname

Structure cleavage enzymes

Occurrence

Kestosis
6-kestosis
Melicitis
Planteose

Tetrasaccharides

GIu 1/2 Fru 1/2 Fru

GIu 1/2 Fru 6/2 Fru

GIu 1/3 Fru 2/1 GIu

Gal 1/6 Fru 1/2 GIu

-Glucosidase

Fungi tetraosis GIu 1/2 Fru 1/2 Fru 1/2 Fru 1/6 Gal 1/6 Gal 1/6 GIu Stachyosis Gal 1/6 Gal 1/6 GIu 1/2 Fru Fru O Lychnosis Gal 1/6 GIu 1/2 Fru 1/1 Gal co
Q Bisfurcose To- - (2/1 Fru 2/6 Fru) - O Fructofuranosysucrose OO * ■ * Pentaoses O Verbascosis Gal on 1/2 «J Polysaccharides

Invertase

Inulin

Irisine

Sitosin

Laevane

Fru 1/2 / Fru 1/27 2/1 GIu

(plus approx. 6% glucose units in the chain) invertase
Inulase

Fru 2/6 / Fru 2/67 Fru 2/1 GIu

Bee honey
Bee honey

manna
tobacco

Artichokes, soy

oats

Wollkraut / Wur zeIn

Dahlia bulbs

chicory

artichokes

Orris roots

Ears of wheat

Grasses, stalks

cn <s \

TABLE (continued)

Surname

structure

Cleavage enzymes

Occurrence

Phlein
Avenarin
Secalin
Pructosan

Fru 2/6, / Fru 2 / β7 _η Fru 2/1 GIu

Branched polyfructoside / 2/6 linkage7 with terminal glucose

Oat straws pogging straw

O O CD

cn cn co

Another object of the invention is a reagent for the determination of fructose and fructose-containing Glycosides, which is characterized in that it is UDPG, sucrose synthetase, buffer substance and a system for determining UDP.

In a preferred embodiment, this reagent consists the system for the determination of UDP from phosphoenolpyruvate, pyruvate kinase, lactate dehydrogenase and NADH.

Particularly favorable results using this preferred system are obtained with a reagent which ο

o, o5 to 1o U / ml sucrose synthetase, 0.05 to 50 U / ml pyruvate kinase, 0.05 to 50 U / ml lactate dehydrogenase, 1 to 15 mmol / 1 UDPG,

0.1 to 1o mmol / 1 phosphoenolpyruvate, 0.1 to 1 mmol / 1 NADH and Contains 0.05 to 0.5 mol / l buffer, pH 6 to 10.5, based on the finished test solution.

The above concentration data relate to the finished test reagent in dissolved form. In addition to the listed Components, the reagent can also contain magnesium salts, stabilizers, activators for the enzymes. Such substances are known to the person skilled in the art and are customary for enzymatic reagents. The reagent lies preferably in dry form, for example in lyophilized State, as it is more durable in this form than in the dissolved state. The reagent have a solid, preferably sheet-shaped or strip-shaped carrier which is impregnated with the reagent is to form a so-called "test strip". The ones used for test strips are suitable

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common carriers, such as paper, porous plastic films, etc.

In another embodiment, the system exists for the determination of UDP from a phosphate donor, e.g. ATP, formyl phosphate or arginyl phosphate, the corresponding Kinase, e.g. B. ATP kinase, formate kinase or arginine kinase, and a reagent for determining the dephosphorylated phosphate donor.

The invention is basically suitable for the rapid and simple, trouble-free determination of any fructose Origin. The method is particularly suitable for determining fructose in biological substances such as Foods, body fluids and the like. A A particular advantage is that the determination can also be carried out in the presence of large excesses of other carbohydrates is feasible.

The following examples further illustrate the invention.

The abbreviations used in the examples and in the preceding description are in the following list summarized and explained:

GOD glucose oxidase

LDH lactate dehydrogenase

NAD ß-nicotinamide adenine dinucleotide

NADH ß-nicotinamide adenine dinucleotide, reduced

PEP phosphoenolpyruvate

PK pyruvate kinase

Tra triethanolamine

UDP uridine 5'-diphosphate

UDPG uridine-5'-diphosphoglucose

UTP uridine 5'-triphosphate.

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example 1

General determination of fructose from aqueous solution: measuring temperature: 25 ° C, measuring wavelength: 36 5 nt, 1 cm cuvettes, test volume: 3.12 ml.

Initial solution

Sample blank concentration (ml) (ml) in the test

Tra buffer, pH 8.0; 0 , 1 mol / l 1, 0 1, o 2 o, 2 33 mmol / l NADH, 6.0 mmol / 1 0, 1 o, 1 0.2 mmol / l PEP, 45 mmol / 1 0, 1 o, 1 1.5 mmol / l UDPG, 73 mmol / 1 0, 1 o., 1 2.4 mmol / l MgCl ₂ and KCl, each o, 2 Minor 0, 1 o, 1 6.7 mmol / l each PK / LDH, 2oo U / ml each 0, o2 o, o2 1.3 U / ml each H ₂ O 1, 4th 1.5 - Sample solution (approx. 0.5
Fructose / ml) mg 0, ¹ ca 0.1 mmol / l mix, absorbance E ₁ after approx. 5 Minutes measure, then start with Sucrose synthetase,
(from wheat germ) 2o U / ml 0, 1.3 U / ml

Mix, incubate for approx. 30 minutes, after the decrease in absorbance has stopped, measure the _{absorbance of the sample and the blank value (E 2).} Calculation of Δ E from the extinction differences. The fructose content of the sample results from the difference according to the formula:

Fructose ßMol / XJ = Δε χ 9.18

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Example 2

Determination of sucrose in pure solution. Measuring temperature: 25 C, measuring wavelength: 365 nm, 1 cm cuvettes, test volume: 3.34 ml.

Initial solution

Sample blank Concentration (ml) (ml) in the incubation solution or in the test

Citrate buffer, ο, 32 mol / l ,.
pH 4.6 o, 2 , 02 o, 2 0, 2 mol / l O ₁ . Encore from mmol / 1 Invertase (ß-fructosidase)
80 U / ml 0 ,1 o, o2 5 U / ml O ₁ I / o 89 18 mmol / l Sample solution (approx. 1 mg sac
charose / ml) 0 approx ι 1 mmol / 1 0, o, 1 0, 3 mmol / l mix for about 15 minutes Incubate at room temperature, O ₁ o, 1 1, 2 nmoles / 1 then heat for 2 minutes at 100 ° C, O, o, 1 2, 0 mmol / 1 Tra buffer, pH 8.0,
0.3 mol / l 1, o, 1 6 / 1.2 U / ml NADH, 6.0 mmol / 1 approx. o, o2 ever - PEP, 45 mmol / 1 1/5 measure, then UDPG, 73 mmol / 1 O ₁ Minutes MgCl ₂ and KCl, each 0.2 mol / l , 0 PK / LDH, each 200 U / ml ,1 o, 2 H ₂ O _r 1 2 U / ml mix, add absorbance E ₁ ,1 start with ,1 Sucrose synthetase 2o. U / ml , 02 , 4 , 5 , 2

Mix, incubate for approx. 30 minutes, after the decrease in absorbance has stopped, measure the absorbance of the sample against the absorbance of the blank value (E ₂ ). Calculation of TU from the absorbance differences. The sucrose content results from the difference according to the formula:

Sucrose ^ in mol / lJ = Δε χ 9.82

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Example 3.

Determination of low fructose concentrates in addition to 200-fold excess of glucose concentrates. Measuring temperature: 25 ° C, measuring wavelength: 365 nm, 1 cm cuvettes, test volume: 3.12 ml.

From the very beginning

Sample blank concentration (ml) (ml) in the test

Tra buffer, pH 8, o; o, 1 mol / l 1, o

NADH, 6, ο mmol / 1 'o, 1

PEP, 45 mmol / 10, 1

UDPG, 73 mmol / 10, 1

MgCl ₂ / KCl, each o, 2 mol / lo, 1

PK / LDH, each 2oo U / ml o, o2

Glucose, 32o mmol / 10, 1

Sample solution (approx. 0.5 mg
Fructose / ml)

1.3

1, ο 33 mmol / 1 o, 1 O, 2 mmol / l o, 1 1, 5 mmol / l o, 1 2, 4 mmol / l o, 1 ever 6.7 mmol / l o, o2 ever 1.3 U / ml o, 1 1o mmol / 1 1.4 -

approx. 0.1 mmol / 1

mix, absorbance
start with

measure after about 5 minutes, then

Sucrose synthetase, 2ο U / ml o, 2 o, 2

1.3 U / ml

Mix, incubate for approx. 30 minutes, after the decrease in absorbance has stopped, measure the absorbance of the sample and the absorbance of the blank value (E ₂ ).

Calculation of Δε from the extinction differences. The fructose content of the sample results from the difference according to the formula:

Fructose / mmol / l / = Δ.Ε χ 9.18

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Example 4.

Determination of inulin in serum. By hydrolysis with acid, inulin is quantitatively hydrolyzed to fructose, which is then determined. Hydrolysis temperature: 80 ⁰ C, measurement temperature: 25 ° C, measurement wavelength: 36 5 nm, 1 cm cuvette, test volume: 3.22 ml.

Initial solution

Sample blank concentration (ml) (ml) in the incubator. solution

Whole blood (with approx. 0.5 g inulin / 1)

Perchloric acid, 0.3 mol / l

0.1 - approx. Ο / 28 mmol / 1 1, o 1, o o, 27 mol / l

Mix and add about 15 minutes approx . Hold 80 ⁰ C, cool down 0, 1 5 2 concentration o, 1 and centrifuge. Remove from centrifugate: 1, 0 in the test 1.093 mmol / l Or 1 0.19 mmol / 1 O/ 1 0.1 2.3 mmol / l got over 0, 1 0.1.5 mmol / l Tra buffer, pH 8.0; 0.3 moles 0, 1 0.16.2 mmol / l NADH, 6.0 mmol / 1 0, o2 o, o2 1.3 U / ml UDPG, 73 mmol / 1 1-, 4th 1.5 - PEP, 45 mmol / 1 approx. Measure minutes, then MgCl ₂ / KCl each 0.2 mol / l PK / LDH each 200 U / ml 0, 0.2 U / ml H ₂ O mix, absorbance E ,. after start with Sucrose synthetase, 2o U / ml

Mix, incubate for approx. 30 minutes, after the decrease in absorbance has stopped, absorbance of the sample and the blank value

(E-) measure.

£ 0

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Calculation of ΔE from the extinction differences » The difference is the inulin content after

Formula:

inulin / roMol / 1 / = Δε χ 3 _f 22> (1, ο + ο, Ι.Q, 8T ^a) ) .1, o4 ^b >

J f fs O ψ \

= Δε χ Ιο, 65

a) Factor calculated from the volume contraction of the blood after deproteinization.

b) Factor, which the average glxicose content of 4% taken into account in inulin.

If free fructose is present in the blood, this must be carried out in a separate test batch without perchloric acid treatment to be determined.

The other reaction conditions correspond to those of Example 1.

ORIGINAL INSPiCTEÖ

Claims (10)

Claims 1J Method for the determination of fructose and fructose containing glycosides, especially in the presence of other sugars or polysaccharides in excess, characterized in that the optionally first from the glycoside in freedom Set fructose is reacted with UDPG and sucrose synthetase to form UDP, which in itself is determined in a known manner. 2. The method according to claim 1, characterized in that the implementation with UDPG at a pH between 6 and 10.5 is carried out. 3. The method according to claim 1 or 2, characterized in that the fructose-containing Glycoside sucrose is determined and the fructose is released from it by invertase. 4. The method according to any one of claims 1 to 3, characterized in that containing fructose Glycoside inulin is determined and the fructose is released from it by acid hydrolysis. 5. The method according to any one of the preceding claims, characterized in that UDP formed by reaction with phosphoenolpyruvate, pyruvate kinase and lactate dehydrogenase is carried out in the presence of NADH and the decrease in NADH is measured. 6. Reagent for the determination of fructose and fructose-containing glycosides, thereby marked zei chn e t that it contains UDPG, sucrose synthetase, buffer substance and a system for determining UDP. 030064/0157 ORIGINAL INSPECTED 7. reagent according to claim 6, characterized in that the system for determining UDP from phosphoenolpyruvate ,. Pyruvate kinase, lactate dehydrogenase, and NADH. 8. reagent according to claim 7, characterized in that it 0.05 to 10 U / ml sucrose synthetase, 0.05 to 50 U / ml pyruvate kinase, 0.05 to 50 U / ml lactate dehydrogenase, 1 to 15 mol / 1 UDPG, 0.1 to 1o mmol / 1 phosphoenolpyruvate, 0.1 to 1 mmol / 1 NADH and Contains 0.05 to 0.5 mol / l buffer, pH 6 to 10.5, based on the finished test solution. 9. reagent according to claim 6, characterized in that the system for determining UDP from a phosphate donor, its specific kinase and a reagent for the determination of the dephosphorylated Phosphate donor. 10. Reagent according to one of claims 6 to 9, characterized characterized in that it also contains magnesium salt and / or stabilizers and / or activators or / and contains a solid sheet or strip-shaped carrier material. 03006A / 0157